TaqMan Fast Advanced Master Mix - assets.thermofisher.cn

For Research Use Only. Not for use in diagnostic procedures.

TaqMan™ Fast Advanced Master MixUSER GUIDE

For two-step real-time RT-PCR in gene expression experimentsor quantitative analysis

Catalog Numbers 4444556, 4444557, 4444558, 4444963, 4444964, 4444965

Publication Number MAN0025706

Revision A.0

Thermo Fisher Scientific Baltics UAB | V.A. Graiciuno 8, LT-02241 | Vilnius, LithuaniaFor descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BELIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH ORARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0025706

Revision Date Description

A.0 9 November 2021 Pub. No. 4444605 Rev. D was converted to Pub. No. MAN0025706Rev. A.0, with the following changes:

• Updated storage temperatures.

• Added QuantStudio™ 6 Pro and 7 Pro Real-Time PCR Systems.

• Added information about recommended algorithms for dataanalysis.

• Added instructions to dilute 60X assays, where applicable.

• Added instructions to divide 20X assays before freezing to avoidmultiple freeze‑thaw cycles.

• Added algorithm recommendations for data analysis.

• Updated polymerase activation time for TaqMan™ Gene ExpressionAssays (single‑tube and TaqMan™ Array Plates).

• Updated thermal cycling conditions for TaqMan™ Gene ExpressionAssays and Custom TaqMan™ Gene Expression Assays—single-tube assays.

• Updated PCR guidelines for TaqMan™ Gene Expression Assays—TaqMan™ Array Cards.

• Updated anneal/extend temperature for TaqMan™ Gene ExpressionAssays and TaqMan™ Array Cards on the 7900HT Fast Real-TimePCR Instrument.

• Removed ramp rates from thermal cycling conditions for TaqMan™

Gene Expression Assays—TaqMan™ Array Cards.

• Removed use of template files to set up PCR reactions forTaqMan™ Gene Expression Assays—TaqMan™ Array Cards. Addedimport of setup files and thermal cycling conditions for this format.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of theseproducts, you accept the terms and conditions of all applicable Limited Use Label Licenses.

Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan isa trademark of Roche Molecular Systems, Inc., used under permission and license. Microsoft and Excel are trademarks of MicrosoftCorporation. AmpliTaq is a registered trademark of Roche Molecular Systems, Inc.

©2021 Thermo Fisher Scientific Inc. All rights reserved.

https://www.thermofisher.com/symbols-definition

Contents

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Purpose of this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

■ CHAPTER 2 RT–PCR for TaqMan™ and Custom TaqMan™ GeneExpression Assays—single–tube assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Perform reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Perform real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Prepare the PCR Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Prepare the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Set up a plate document or plate file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Run the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Analyze data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Algorithms for data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

■ CHAPTER 3 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17


Perform real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Prepare the PCR Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Prepare the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Set up a plate document or plate file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Run the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20


TaqMan™ Fast Advanced Master Mix User Guide 3

■ CHAPTER 4 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array Cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22


Perform real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Recommended amount of cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Prepare the sample–specific PCR Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Prepare the TaqMan™ Array Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Set up a card document or card file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Run the TaqMan™ Array Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24


■ CHAPTER 5 RT–PCR for TaqMan™ MicroRNA Assays—single–tube assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26


Perform real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Prepare the PCR Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Prepare the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Set up a plate document or experiment file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Run the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29


■ CHAPTER 6 RT–PCR for TaqMan™ Advanced miRNA Assays—single–tube assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31


Perform real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Prepare PCR Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Prepare the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Set up a plate document or experiment file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Run the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34


Contents

4 TaqMan™ Fast Advanced Master Mix User Guide

■ APPENDIX A Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Components of the TaqMan™ Fast Advanced Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . 36AmpliTaq™ Fast DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Uracil-N glycosylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36dUTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36ROX™ Passive Reference dye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Two–step real-time RT–PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

About the 5' nuclease assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

TaqMan™ MGB probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Enzyme activation time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

■ APPENDIX B Best practices for PCR and RT-PCR experiments . . . . . . . . . . . . . . 40

Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Use UNG to prevent false-positive amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Detect fluorescent contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

■ APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

■ APPENDIX D Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Contents


Product information

Product descriptionThe Applied Biosystems™ TaqMan™ Fast Advanced Master Mix enables real-time PCR in any geneexpression experiment or quantitative analysis, including the following applications:

• Pathogen detection

• Differential gene expression analysis

• Viral load quantitation

• MicroRNA quantitation

• Microarray verification

The Master Mix can be used with any DNA target, including complementary DNA (cDNA) or genomicDNA (gDNA). It can be used in the second step of a two-step real-time RT–PCR protocol for RNAquantitation experiments. A cDNA template can be generated from RNA using one of our reversetranscription kits prior to real-time PCR with the Master Mix. See details in “Required materials notsupplied” on page 7.

The Master Mix is supplied at a 2X concentration and contains the following components:

• AmpliTaq™ Fast DNA Polymerase

• Uracil-N glycosylase (UNG)

• dNTPs with dUTP

• ROX™ Reference Dye (passive reference)

• Optimized buffer components

For more information about each component, see “Components of the TaqMan™ Fast Advanced MasterMix” on page 36.

The Master Mix is optimized for use with primers and TaqMan™ probes designed according to ourguidelines.

Purpose of this guideThis document describes how to perform two-step real-time RT-PCR using TaqMan™ Fast AdvancedMaster Mix with the following assays or components:

• TaqMan™ Gene Expression Assays (single-tube assays, array plates, and array cards)– Single‑tube assays

– TaqMan™ Array Plates

– TaqMan™ Array Cards

• Custom TaqMan™ Gene Expression Assays

1


• TaqMan™ MicroRNA Assays

• TaqMan™ Advanced miRNA Assays

This guide provides general guidelines for analyzing data. Analysis can vary between applications. Formore information about procedures and data analysis, see the documentation for your instrument.

Contents and storageTable 1 TaqMan™ Fast Advanced Master Mix

Cat. No. Number of 20-µL reactions Amount Storage[1]

4444556 100 1 × 1 mL

2–8°C

4444557 500 1 × 5 mL

4444963 (2 × 4444557) 1,000 2 × 5 mL

4444964 (5 × 4444557) 2,500 5 × 5 mL

4444965 (10 × 4444557) 5,000 10 × 5 mL

4444558 5,000 1 × 50 mL

[1] See label for expiration date.

Required materials not suppliedUnless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates thatthe material is available from fisherscientific.com or another major laboratory supplier.

Table 2 Instrument, software, equipment, plates and accessories, and consumables

Item Source

Instrument, one of the following:

QuantStudio™ 6 Pro Real-Time PCR System[1]

Contact your local sales office.

QuantStudio™ 7 Pro Real-Time PCR System

QuantStudio™ 3 and 5 Real-Time PCR Instruments[1]

QuantStudio™ 6 Flex Real-Time PCR System[1]

QuantStudio™ 7 Flex Real-Time PCR System

QuantStudio™ 12K Flex Real–Time PCR System

StepOne™ Real-Time PCR System[2]

StepOnePlus™ Real-Time PCR System[1]

7500 Real-Time PCR System[1]

Chapter 1 Product informationContents and storage 1


https://www.thermofisher.com/search/results?query=4444556&focusarea=Search%20All&scope=PDF









http://www.thermofisher.com

http://fisherscientific.com

Table 2 Instrument, software, equipment, plates and accessories, and consumables (continued)

Item Source

7500 Fast Real-Time PCR System[1]

Contact your local sales office.

ViiA™ 7 Real-Time PCR System

7900HT Real-Time PCR Instrument[1]

7900HT Fast Real-Time PCR Instrument

Compatible real–time PCR instruments from other suppliers may beacceptable. Verify thermal–cycling conditions on other real–time PCRinstruments.

Software

Microsoft™ Excel™ (Optional, to create plate layout files for import) microsoft.com

Equipment

Centrifuge with plate adapter MLS

Microcentrifuge MLS

Thermal cycler, or heat block or water bath set to 95°C MLS

Adjustable pipettors MLS

Laboratory mixer (vortex or equivalent) MLS

Tubes, plates, and other consumables

Plastics consumables thermofisher.com/plastics

Pipette tips thermofisher.com/pipettetips

Disposable gloves MLS

[1] Not compatible with TaqMan™ Array Cards.[2] Not compatible with TaqMan™ Array Plates or TaqMan™ Array Cards.

Table 3 Reagents for reverse transcription

Item Source

Reagents for reverse transcription (all assays)

TE, pH 8.0, RNase-free AM9849

(Optional) RNase inhibitorN8080119

AM2684 (Cloned; 40 U/µL)

Nuclease-Free Water (not DEPC-Treated) AM9930

Chapter 1 Product informationRequired materials not supplied1


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http://www.thermofisher.com/plastics

http://www.thermofisher.com/pipettetips

https://www.thermofisher.com/search/results?query=AM9849&focusarea=Search%20All&scope=PDF

https://www.thermofisher.com/search/results?query=AM9930&focusarea=Search%20All&scope=PDF

Table 3 Reagents for reverse transcription (continued)

Item Source

Reagents for reverse transcription (TaqMan™ Gene Expression Assays)

High-Capacity cDNA Reverse Transcription Kit4368814

4374966 (with RNase inhibitor)

High-Capacity RNA-to-cDNA™ Kit 4387406

SuperScript™ VILO™ cDNA Synthesis Kit 11754050

SuperScript™ IV VILO™ Master Mix 11756050

Reagents for reverse transcription (TaqMan™ MicroRNA Assays)

TaqMan™ MicroRNA Reverse Transcription Kit[1] 4366596

TaqMan™ Advanced miRNA Assays

TaqMan™ Advanced miRNA cDNA Synthesis Kit[2] A28007

[1] TaqMan™ MicroRNA Assays are optimized for use with the TaqMan™ MicroRNA Reverse Transcription Kit. Assay performance cannot be guaranteed with other reverse transcription kits.

[2] TaqMan™ Advanced miRNA Assays are optimized for use with the TaqMan™ Advanced miRNA cDNA Synthesis Kit. Assay performance cannot be guaranteed with other reverse transcription kits.

Table 4 Assays

Item Source

TaqMan™ Assays

TaqMan™ Gene Expression Assays thermofisher.com/taqmangeneexpression

Custom TaqMan™ Gene Expression Assays thermofisher.com/taqmancustomgeneexpression

Custom TaqMan™ probes and primers[1] thermofisher.com/customprimersprobes

TaqMan™ Array Plates

96‑well Fast (0.1‑mL) TaqMan™ Array Plates and96‑well Standard (0.2‑mL) TaqMan™ Array Plates

thermofisher.com/taqmanarrays

TaqMan™ Array Cards

TaqMan™ Array Card thermofisher.com/taqmanarrays

TaqMan™ MicroRNA Assays

TaqMan™ MicroRNA Assays thermofisher.com/taqmanmirna

Custom TaqMan™ Small RNA Assays thermofisher.com/taqmancustommirna

TaqMan™ Advanced miRNA Assays

TaqMan™ Advanced miRNA Assays thermofisher.com/advancedmirna

[1] Synthesized to your sequence and choice of quencher and reporter dyes.

Chapter 1 Product informationRequired materials not supplied 1




https://www.thermofisher.com/search/results?query=A28007&focusarea=Search%20All&scope=PDF

http://www.thermofisher.com/taqmangeneexpression

http://www.thermofisher.com/taqmancustomgeneexpression

http://www.thermofisher.com/customprimersprobes

http://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/taqman-gene-expression/real-time-pcr-taqman-arrays.html?CID=fl-WE112784

http://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/taqman-gene-expression/real-time-pcr-taqman-arrays.html?CID=fl-WE112784

http://www.thermofisher.com/taqmanmirna

https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/mirna-ncrna-taqman-assays/custom-taqman-small-rna-assays.html

http://www.thermofisher.com/advancedmirna

Table 5 Kits and reagents for RNA isolation

Item Source

RNA isolation products thermofisher.com/rnaisolation

Supporting reagents thermofisher.com/rnaisolationreagents

Chapter 1 Product informationRequired materials not supplied1


http://thermofisher.com/rnaisolation

http://thermofisher.com/rnaisolationreagents

Workflow

Perform reverse transcription

▼

Perform real-time PCR amplification

Prepare the PCR Reaction Mix

▼

Prepare the PCR reaction plate or card

▼

Set up a plate or card document, or experiment file

(or use the document provided with the cards or custom plates)

▼

Run the PCR reaction plate or card

▼

Analyze the data

Chapter 1 Product informationWorkflow 1


RT–PCR for TaqMan™ and CustomTaqMan™ Gene Expression Assays

—single–tube assays

Perform reverse transcriptionPerform reverse transcription to obtain cDNA from RNA samples.

For information on reverse transcription kits, see Table 3 on page 8. For detailed guidelines andinstructions see TaqMan™ Gene Expression Assays User Guide—single-tube assays (Pub. No. 4333458).

Perform real-time PCR

Guidelines

• Store the assays frozen.

• Protect from light until use. Excessive exposure to light might affect the fluorescent probes.

• Multiple assays can be run on one reaction plate. Include no-template controls (NTCs) for eachassay.

Before you begin

Dilute 60X assays to 20X working stocks with TE, pH 8.0, RNase-free.

• Divide the solutions into smaller aliquots to minimize freeze–thaw cycles.

Note: The aliquot size depends on how many PCR reactions you will run.

• Determine the total number of PCR reactions required, including replicates, for each sample.Include a no-template control for each assay.

Note: We recommend four replicate reactions for each assay.

• Thaw the Master Mix on ice, then mix thoroughly but gently.

• Thaw the assays on ice, then vortex and briefly centrifuge to resuspend.

• Thaw the samples on ice, then vortex and briefly centrifuge to resuspend.

2



1. Combine the following components in the quantities shown, multiplied by the number of reactionsrequired.

Add 10% overage for pipetting loss. The minimum final dilution of RT Reaction Mix in PCRReaction Mix is 1:15.

Component

Volume per reaction

Final concentration384–well, 96–wellfast (0.1–mL) plates

96–well standard(0.2–mL), 48–well

plates

TaqMan™ Fast Advanced MasterMix (2X)

5.0 µL 10.0 µL 1X

TaqMan™ Assay (20X) 0.5 µL 1.0 µL 1X

Nuclease-free water[1] 3.5 µL 7.0 µL —

Total volume of PCR ReactionMix per reaction

9.0 µL 18.0 µL —

[1] Adjust the volume of nuclease–free water for a larger volume of cDNA.

2. Vortex briefly to mix.

3. Centrifuge briefly to bring the PCR Reaction Mix to the bottom of the tube.

Prepare the PCR reaction plate

1. Transfer the appropriate volume of PCR Reaction Mix to each well of the plate.

2. Add cDNA template (1 pg to 100 ng in nuclease–free water), or nuclease–free water for NTC, toeach well.

• 384-well plate, 96–well fast (0.1 mL) plate: 1.0 µL

• 96–well standard (0.2 mL) plate, 48–well plate: 2.0 µL

Note: Adjust the volume of nuclease–free water in the PCR Reaction Mix for a larger volume ofcDNA.

3. Seal the reaction plate with optical adhesive film, then centrifuge briefly to bring the PCR ReactionMix to the bottom of the well.

4. Apply a compression pad to the plate, if required by your real-time PCR system.

Chapter 2 RT–PCR for TaqMan™ and Custom TaqMan™ Gene Expression Assays—single–tube assaysPerform real-time PCR 2


Set up a plate document or plate file

See the appropriate instrument user guide for detailed instructions to program the thermal-cyclingconditions or to run the plate.

Note: The instrument must be configured with the block appropriate for the plate type.

1. Set up a plate document or plate file using the following conditions.

Real–time PCR system

(Optional) UNGincubation

Polymeraseactivation[1] PCR (40 cycles)

Hold

50°C

Hold

95°C

Denature

95°C

Anneal / extend

60°C

• QuantStudio™ 6 Pro and 7 ProReal-Time PCR Systems

• QuantStudio™ 3/QuantStudio™ 5Flex Real‑Time PCR System

• QuantStudio™ 6 / QuantStudio™

7 Flex Real-Time PCR System

• QuantStudio™ 12K Flex Real–Time PCR System

• StepOne™ Real-Time PCRSystem

• StepOnePlus™ Real-Time PCRSystem

• ViiA™ 7 Real-Time PCR System

• 7900HT Real-Time PCR System

• 7900HT Fast Real‑Time PCRSystem

2 minutes 20 seconds[2] 1 second 20 seconds

• 7500 Fast Real-Time PCRSystem

• 7500 Real-Time PCR System2 minutes 20 seconds[2] 3 seconds 30 seconds

[1] To activate AmpliTaq™ Fast DNA Polymerase.[2] Enzyme activation can continue for up to 2 minutes without affecting the results. See “Enzyme activation time” on page 39.

2. Select the appropriate block, if this option applies to your instrument.

3. Select the appropriate experiment type, if this option applies to your instrument.

4. Select TaqMan™ Reagents to detect the target sequence, if this option applies to your instrument.

Chapter 2 RT–PCR for TaqMan™ and Custom TaqMan™ Gene Expression Assays—single–tube assaysPerform real-time PCR2


5. Select a run mode.

Real-time PCR system Run mode

• 7500 Real-Time PCR System


• 7900HT Fast Real‑Time PCR System (384–well and 96–well standardblock modules)

Standard

• QuantStudio™ 6 Pro and 7 Pro Real-Time PCR Systems

• QuantStudio™ 3/QuantStudio™ 5 Flex Real‑Time PCR System

• QuantStudio™ 6 / QuantStudio™ 7 Flex Real-Time PCR System


• StepOne™ Real-Time PCR System

• StepOnePlus™ Real-Time PCR System


• 7500 Fast Real-Time PCR System


Fast

6. Enter the sample volume, if this option applies to your instrument.



Run the PCR reaction plate

1. Open the plate document or experiment file that corresponds to the reaction plate in the systemsoftware.

2. Load the reaction plate.

3. Start the run.

Analyze dataData analysis varies depending on your real–time PCR system. See the instrument user guide for moreinformation.

1. View the amplification plots for the reactions.

2. Use auto baseline and auto threshold settings, or set the baseline and threshold values todetermine the threshold cycles (Ct) for the amplification curves.

3. Use the relative standard curve method or the comparative Ct method to analyze data.

Chapter 2 RT–PCR for TaqMan™ and Custom TaqMan™ Gene Expression Assays—single–tube assaysAnalyze data 2


Algorithms for data analysis

Table 6 Algorithm recommendations for single–tube assays

Algorithm Recommendation

Threshold (Ct) Recommended.

Relative threshold (Crt) (Optional) Use for troubleshooting abnormal or unexpected results.

The relative threshold algorithm is available in the Relative Quantification application on Thermo Fisher™

Connect (thermofisher.com/connect).

Chapter 2 RT–PCR for TaqMan™ and Custom TaqMan™ Gene Expression Assays—single–tube assaysAnalyze data2


https://www.thermofisher.com/connect

RT–PCR for TaqMan™ GeneExpression Assays—TaqMan™ Array

Plates


For information on reverse transcription kits, see Table 3 on page 8. For detailed guidelinesand instructions see TaqMan™ Gene Expression Assays User Guide—TaqMan™ Array Plates (Pub.No. 4391016).


Guidelines

Store the plates away from light until use. Excessive exposure to light might affect the fluorescentprobes.

Before you begin

• Determine the total number of PCR reactions.

One reaction corresponds to one well in the plate.



3



1. Combine the following components for the number of reactions required.

Add 10% overage for pipetting loss.

Component

Volume per reaction

96–well fast (0.1–mL) plate96–well standard (0.2–mL)

plate

cDNA template + Nuclease-free water[1] 5 µL 10 µL

TaqMan™ Fast Advanced Master Mix (2X) 5 µL 10 µL

Total volume of PCR Reaction Mix perreaction

10 µL 20 µL

[1] 5–50 ng of cDNA diluted in nuclease–free water.




1. Transfer the appropriate volume of PCR Reaction Mix to each well of the plate.

• 96–well fast (0.1 mL) plate: 10 µL

• 96–well standard (0.2 mL) plate: 20 µL


2. Seal the plate with optical adhesive film, then centrifuge briefly to bring the PCR Reaction Mix tothe bottom of the wells.


Chapter 3 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array PlatesPerform real-time PCR3


Set up a plate document or plate file



1. Import the setup file (SDS in TXT format) into the real-time PCR instrument or software.





Hold

50°C

Hold

95°C

Denature

95°C

Anneal / extend

60°C

• QuantStudio™ 6 Pro and 7 ProReal-Time PCR Systems

• QuantStudio™ 3/QuantStudio™ 5Flex Real‑Time PCR System

• QuantStudio™ 6 / QuantStudio™

7 Flex Real-Time PCR System


• StepOnePlus™ Real-Time PCRSystem



• 7900HT Fast Real‑Time PCRSystem

2 minutes 20 seconds[2] 1 second 20 seconds

• 7500 Fast Real-Time PCRSystem

• 7500 Real-Time PCR System2 minutes 20 seconds[2] 3 seconds 30 seconds

[1] To activate AmpliTaq™ Fast DNA Polymerase.[2] Enzyme activation can continue for up to 2 minutes without affecting the results. See “Enzyme activation time” on page 39.




Chapter 3 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array PlatesPerform real-time PCR 3






• 7900HT Fast Real‑Time PCR SystemStandard









Fast


• 96–well fast (0.1 mL) plate: 10.0 µL

• 96–well standard (0.2 mL) plate: 20.0 µL




3. Start the run.



2. Set the baseline and threshold values to determine the threshold cycles (Ct) for the amplificationcurves, or select relative threshold under analysis settings to obtain (Crt) values.


Chapter 3 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array PlatesAnalyze data3



Table 7 Algorithm recommendations for TaqMan™ Array Plates


Threshold (Ct) • Recommended for data analysis.

Relative threshold (Crt)

• (Optional) Use for data analysis.

• Use to troubleshoot unexpected results.

• Use to correct a variable baseline, which can be due to dried-downassays on the plate being reconstituted at different rates.



Chapter 3 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array PlatesAnalyze data 3



RT–PCR for TaqMan™ GeneExpression Assays—TaqMan™ Array

Cards


For reverse transcription kits see Table 3 on page 8. For detailed guidelines and instructions see theprotocol for your kit and the TaqMan™ Gene Expression Assays User Guide—TaqMan™ Array Cards(Pub. No. 4400263) .


Guidelines

• Store the card in its packaging until the packaging has reached room temperature and you areready to fill it with sample–specific PCR Reaction Mix.

• Protect from light. Prolonged exposure to indoor lighting can degrade the fluorescent probes in thecard. Do not expose the card to sunlight.

• Fill each fill reservoir with sample–specific PCR Reaction Mix made from a single cDNA sample.

• Use 100 µL of sample–specific PCR Reaction Mix to fill each fill reservoir. Volumes smaller than 100µL will result in insufficiently filled cards.

• Do not add the sample after centrifuging the cards. Centrifugation of the card causes the sample–specific PCR Reaction Mix to resuspend the dried TaqMan™ probes and primers within the wells ofthe card. Addition of the sample after centrifuging disrupts the resuspended assay positions.

• After loading the card with PCR Reaction Mix equilibrate the card to room temperature beforeloading it into the real–time PCR instrument.

• Run the card within 72 hours of sealing it.

• Protect the card from light and store at 2–8°C if a run is not started immediately after sealing.

4


Recommended amount of cDNA

• We recommend 30–1000 ng (0.3–10 ng/µL) of cDNA (converted from total RNA) per fill reservoir.

• The amount of cDNA to use depends on the expression level of the target genes and the number oftarget copies per well that need to be detected. For example:

– Use 1000 ng (10 ng/µL) per fill reservoir to detect genes with low expression.Because the cDNA concentration is high, use high-quality cDNA without inhibitors.

– Use 100–200 ng per fill reservoir to detect genes with moderate expression.

– Use 30–50 ng per fill reservoir to detect genes with moderate to high expression.

• Use the same amount of cDNA sample for all reactions.

Before you begin

• Determine the number of fill reservoirs in the card that will be used for each cDNA sample.



Prepare the sample–specific PCR Reaction Mix



Component Volume per fill reservoir

cDNA template + Nuclease-free water[1] 50 µL

TaqMan™ Fast Advanced Master Mix (2X) 50 µL

Total volume 100 µL

[1] See “Recommended amount of cDNA” on page 23.



Prepare the TaqMan™ Array Card

Fill the array card with sample–specific PCR Reaction Mix, then centrifuge and seal the card.

For detailed procedures to prepare the card, see TaqMan™ Gene Expression Assays User Guide—TaqMan™ Array Cards (Pub. No. 4400263).

Chapter 4 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array CardsPerform real-time PCR 4


Set up a card document or card file



1. Import the setup file (TXT format) into the real–time PCR instrument or software.

2. Set up a card document or card file.

Note: Thermal cycling conditions depend on the instrument.

Table 8 ViiA™ 7 Real-Time PCR System and compatible QuantStudio™ systems with fastcycling mode



Hold

50°C

Hold

92°C

Denature

95°C

Anneal / extend

60°C

2 minutes 10 minutes[2] 1 second 20 seconds

[1] To activate AmpliTaq™ Fast DNA Polymerase.[2] To completely dissolve the primers and probes on the card.

IMPORTANT! Do not use the default Fast settings.

Table 9 7900HT Fast Real-Time PCR Instrument with standard cycling mode



Hold

50°C

Hold

92°C

Denature

97°C

Anneal / extend

60°C

2 minutes 10 minutes[2] 1 second 20 seconds

[1] To activate AmpliTaq™ Fast DNA Polymerase.[2] To completely dissolve the primers and probes on the card.

Run the TaqMan™ Array Card

1. Open the card document or experiment file that corresponds to the card in the system software.

2. Load the card.

3. Start the run.

Chapter 4 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array CardsPerform real-time PCR4




2. Set the baseline and threshold values to determine the threshold cycles (Ct) for the amplificationcurves, or select relative threshold under analysis settings to obtain (Crt) values.



Table 10 Algorithm recommendations for TaqMan™ Array Cards


Relative threshold (Crt)

Recommended for the following instruments:

• QuantStudio™ Real‑Time PCR Instruments

• ViiA™ 7 instrument

Can correct a variable baseline, which might be due to dried-downassays on the card being reconstituted at different rates.

Threshold (Ct)Optional if used for analysis of established protocols.

Recommended for 7900HT Fast Real-Time PCR Instrument.



Chapter 4 RT–PCR for TaqMan™ Gene Expression Assays—TaqMan™ Array CardsAnalyze data 4



RT–PCR for TaqMan™ MicroRNAAssays—single–tube assays

This chapter covers use of TaqMan™ MicroRNA Assays. TaqMan™ MicroRNA Assays are also availablein the following formats:

• TaqMan™ Array Cards

• TaqMan™ OpenArray™ Plates

For use of TaqMan™ Advanced miRNA Assays see Chapter 6, “RT–PCR for TaqMan™ Advanced miRNAAssays—single–tube assays”.

See thermofisher.com/taqmanmirna for more information.


Use the TaqMan™ MicroRNA Reverse Transcription Kit. For detailed guidelines and instructions seeTaqMan™ Small RNA Assay User Guide (Pub. No. 4364031).


Guidelines

• Store the assays at –20°C.

• Protect the assays from light until use. Excessive exposure to light might affect the fluorescentprobes.

• Prepare the PCR Reaction Mix before transferring it to the reaction plate for thermal cycling.

Before you begin

• Divide 20X assays into smaller aliquots to minimize freeze–thaw cycles. The size of the aliquotsdepends upon the number of PCR reactions you typically run.

• Determine the total number of PCR reactions required, including a microRNA assay for each cDNAsample, endogenous control assays, and a no-template control (NTC) for each assay.



5


http://www.thermofisher.com/taqmanmirna




Component

Volume per reaction

384–well, 96–well fast(0.1–mL) plates

96–well standard (0.2–mL),48–well plates

TaqMan™ Fast Advanced Master Mix (2X) 5.00 µL 10.00 µL

Nuclease-free water[1] 3.83 µL 7.67 µL

TaqMan™ MicroRNA Assay (20X) 0.50 µL 1.00 µL

cDNA template[2] 0.67 µL 1.33 µL

Total volume of PCR Reaction Mix perreaction

10.00 µL 20.00 µL

[1] Adjust the volume of nuclease–free water for a larger volume of cDNA.[2] The minimum final dilution of RT reaction in PCR reaction is 1:15.

2. Mix gently, then centrifuge to bring the PCR Reaction Mix to the bottom of the tube.


1. Transfer the appropriate volume of PCR Reaction Mix to each well of an optical reaction plate.

• 384-well plate, 96–well fast (0.1 mL) plate: 10 µL

• 48-well plate , 96–well standard (0.2 mL) plate: 20 µL




Chapter 5 RT–PCR for TaqMan™ MicroRNA Assays—single–tube assaysPerform real-time PCR 5


Set up a plate document or experiment file

See the appropriate instrument user guide for detailed instructions to program the thermal–cyclingconditions or to run the plate.





Hold

95°C

Denature

95°C

Anneal / extend

60°C


• QuantStudio™ 3/QuantStudio™ 5 FlexReal‑Time PCR System

• QuantStudio™ 6 / QuantStudio™ 7 FlexReal-Time PCR System

• QuantStudio™ 12K Flex Real–Time PCRSystem





• 7900HT Fast Real‑Time PCR System

20 seconds 1 second 20 seconds


• 7500 Fast Real-Time PCR System20 seconds 3 seconds 30 seconds

[1] To activate AmpliTaq™ Fast DNA Polymerase.




Chapter 5 RT–PCR for TaqMan™ MicroRNA Assays—single–tube assaysPerform real-time PCR5







Standard










Fast







3. Start the run.



2. Use auto baseline and auto threshold settings, or set the baseline and threshold values todetermine the threshold cycles (Ct) for the amplification curves.


Chapter 5 RT–PCR for TaqMan™ MicroRNA Assays—single–tube assaysAnalyze data 5









Chapter 5 RT–PCR for TaqMan™ MicroRNA Assays—single–tube assaysAnalyze data5



RT–PCR for TaqMan™ AdvancedmiRNA Assays—single–tube assays

This chapter covers use of TaqMan™ Advanced miRNA Assays. For use of TaqMan™ MicroRNA Assayssee Chapter 5, “RT–PCR for TaqMan™ MicroRNA Assays—single–tube assays”.

TaqMan™ Advanced miRNA Assays are also available in the following formats:

• TaqMan™ Array Plates

• TaqMan™ Array Cards

• TaqMan™ OpenArray™ Plates(TaqMan™ OpenArray™ Real-Time PCR Master Mix is recommended for TaqMan™ OpenArray™

Plates).

See thermofisher.com/advancedmirna for more information.

For details about predefined TaqMan™ Advanced miRNA Assays see TaqMan™ Advanced miRNAAssays User Guide—TaqMan™ Array Plates (Pub. No. MAN0016120).

Perform reverse transcriptionUse the TaqMan™ Advanced miRNA cDNA Synthesis Kit (Cat. No. A28007). For detailed guidelinesand instructions see TaqMan™ Advanced miRNA Assays User Guide—Single-tube Assays (Pub.No. 100027897).

Prepare cDNA templates with the following reactions:

• Poly(A) tailing reaction

• Adaptor ligation reaction

• Reverse transcription reaction

• miR–Amp reaction


Guidelines

• Store the assays at –20°C.

• Protect the assays from light until use. Excessive exposure to light might affect the fluorescentprobes.

• Prepare the PCR Reaction Mix before transferring it to the reaction plate for thermal cycling.

6


http://www.thermofisher.com/advancedmirna

https://www.thermofisher.com/search/results?query=A28007&focusarea=Search%20All&scope=PDF

Before you begin

• Divide 20X assays into smaller aliquots to minimize freeze–thaw cycles. The size of the aliquotsdepends upon the number of PCR reactions you typically run.

• Determine the total number of PCR reactions required, including a microRNA assay for each cDNAsample, endogenous control assays, and a no-template control (NTC) for each assay.



Prepare PCR Reaction Mix

1. Prepare 1:10 dilutions of the cDNA template.

For example, add 5 µL of the miR-Amp reaction product to 45 µL of 0.1X TE buffer.



Component

Volume per reaction

384–well, 96–well fast (0.1–mL) plates

96–well standard (0.2–mL),48–well plates

TaqMan™ Fast Advanced Master Mix(2X)

5.00 µL 10.00 µL

Nuclease-free water[1] 2.00 µL 4.00 µL

TaqMan™ Advanced miRNA Assay(20X)

0.50 µL 1.00 µL

cDNA template (1:10 dilution) 2.50 µL 5.00 µL

Total volume per reaction 10.00 µL 20.00 µL

[1] Adjust the volume of nuclease–free water for a larger volume of cDNA.

3. Mix gently, then centrifuge to bring the PCR Reaction Mix to the bottom of the tube.


1. Transfer the appropriate volume of PCR Reaction Mix to each well of an optical reaction plate.

• 384-well plate, 96–well fast (0.1 mL) plate: 10 µL

• 48-well plate , 96–well standard (0.2 mL) plate: 20 µL


Chapter 6 RT–PCR for TaqMan™ Advanced miRNA Assays—single–tube assaysPerform real-time PCR6




Set up a plate document or experiment file

See the appropriate instrument user guide for detailed instructions to program the thermal–cyclingconditions or to run the plate.





Hold

95°C

Denature

95°C

Anneal / extend

60°C


• QuantStudio™ 3/QuantStudio™ 5 FlexReal‑Time PCR System

• QuantStudio™ 6 / QuantStudio™ 7 FlexReal-Time PCR System

• QuantStudio™ 12K Flex Real–Time PCRSystem





• 7900HT Fast Real‑Time PCR System

20 seconds 1 second 20 seconds


• 7500 Fast Real-Time PCR System20 seconds 3 seconds 30 seconds

[1] To activate AmpliTaq™ Fast DNA Polymerase.




Chapter 6 RT–PCR for TaqMan™ Advanced miRNA Assays—single–tube assaysPerform real-time PCR 6







Standard










Fast







3. Start the run.



2. Use auto baseline setting and threshold setting of 0.1, or set the baseline and threshold values todetermine the threshold cycles (Ct) for the amplification curves.


Chapter 6 RT–PCR for TaqMan™ Advanced miRNA Assays—single–tube assaysAnalyze data6









Chapter 6 RT–PCR for TaqMan™ Advanced miRNA Assays—single–tube assaysAnalyze data 6



Supplemental information

Components of the TaqMan™ Fast Advanced Master Mix

AmpliTaq™ Fast DNA Polymerase

The AmpliTaq™ Fast DNA Polymerase enzyme is purified through a proprietary process to reducebacterial DNA introduced from the host organism. The purification process ensures that non–specific,false–positive DNA products due to bacterial DNA contamination are minimized during PCR.

When AmpliTaq™ Fast DNA Polymerase is added to the reaction mix at room temperature, the inactiveenzyme is not capable of primer extension. Any low–stringency mispriming events that may haveoccurred will not be enzymatically extended and subsequently amplified. A thermal incubation step isrequired for activation to ensure that active enzyme is generated only at temperatures where the DNA isfully denatured.

Uracil-N glycosylase

Uracil-N glycosylase (UNG) treatment can prevent the reamplification of carryover PCR productsby removing any uracil incorporated into single- or double-stranded amplicons. UNG preventsreamplification of carryover PCR products in an assay if all previous PCR for that assay was performedusing a dUTP-containing master mix. For more information about UNG see “Use UNG to preventfalse-positive amplification” on page 40.

dUTP

This Master Mix includes dUTP to enable uracil-N-glycosylase (UNG) activity and maintain optimal PCRresults.

ROX™ Passive Reference dye

The ROX™ Passive Reference dye provides an internal reference to which the reporter dye signal can benormalized during data analysis. Normalization is necessary to correct for fluorescent fluctuations dueto changes in concentration or volume.

A


Two–step real-time RT–PCRVisit thermofisher.com/qpcreducation for more information.

Note: TaqMan™ MicroRNA Assays and TaqMan™ Advanced miRNA Assays do not use the same RTchemistry.

A target template is a DNA sequence, including cDNA, a gDNA, or a plasmid nucleotide sequence. Anamplicon is a short segment of DNA.

Gene quantification assays using TaqMan™ Fast Advanced Master Mix and TaqMan™ Assays areperformed in a two-step RT–PCR.

1. In the reverse transcription (RT) step, cDNA is reverse transcribed from RNA.

2. In the PCR step, PCR products are quantitatively synthesized from cDNA samples using theMaster Mix.

For details on how the TaqMan™ MGB probe is used in the PCR step, see “TaqMan™ MGB probes” onpage 38.

About the 5' nuclease assayThe 5' nuclease assay process takes place during PCR amplification. It occurs in every cycle and doesnot interfere with the exponential accumulation of product.

During the PCR, the TaqMan™ MGB probe anneals specifically to a complementary sequence betweenthe forward and reverse primer sites.

When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression ofthe reporter fluorescence, primarily by Förster–type energy transfer.

These figures do not represent TaqMan™ Advanced miRNA Assays because the RT chemistry isdifferent. However, the principle of the 5' nuclease assay during PCR amplification remains the same.These assays also use a forward primer, a reverse primer, and a probe.

cDNA template

Target region of cDNA template

Forward primer

Reverse primer

Probe

Reporter dye

Reporter dye, fluorescing

Minor groove binder probe

Non-fluorescent quencher

DNA polymerase

R

R

MGB

NFQ

P

3' 5'5' 3'

Figure 1 cDNA synthesis product

Appendix A Supplemental informationTwo–step real-time RT–PCR A


http://www.thermofisher.com/qpcreducation

3' 5'

5' 3'

R NFQ MGB

Figure 2 Denature and anneal

The DNA polymerase cleaves only probes that hybridize to the target. Cleavage separates the reporterdye from the quencher dye. This results in increased fluorescence by the reporter. The increase influorescence occurs only if the target sequence is complementary to the probe and amplified duringPCR. Because of these requirements, nonspecific amplification is not detected.

Figure 3 Cleavage

Polymerization of the strand continues. However, no extension of the probe occurs during PCR becausethe 3' end of the probe is blocked.

3' 5'

5' 3'

R

Figure 4 Completion of polymerization

TaqMan™ MGB probesTaqMan™ MGB probes contain:

• A reporter dye (for example, FAM™ dye) at the 5′ end of the probe.

• A non-fluorescent quencher (NFQ) dye at the 3′ end of the probe.The NFQ dye does not fluoresce, which allows the real-time PCR system to measure the reporterdye contributions more accurately.

• A minor groove binder (MGB) at the 3´ end of the probe that:– Increases the melting temperature (Tm) without increasing the probe length.

– Allows for the design of shorter probes.

Appendix A Supplemental informationTaqMan™ MGB probesA


Enzyme activation timeUsing TaqMan™ Fast Advanced Master Mix, the enzyme activation step can range from 20 seconds to 2minutes. A 20–second enzyme activation step is sufficient when the template is cDNA. A longer enzymeactivation time will not affect the results.

The enzyme activation time for the default fast thermal cycling conditions on the instruments is 20seconds. If a longer enzyme activation time is required, change the thermal cycling conditions beforestarting the run. A longer enzyme activation time can help to denature double-stranded genomic DNAwhen genomic DNA is used.

Appendix A Supplemental informationEnzyme activation time A


Best practices for PCR and RT-PCRexperiments

Good laboratory practices for PCR and RT-PCR• Wear clean gloves and a clean lab coat.

– Do not wear the same gloves and lab coat that you have previously used when handlingamplified products or preparing samples.

• Change gloves if you suspect that they are contaminated.

• Maintain separate areas and dedicated equipment and supplies for:– Sample preparation and reaction setup.

– Amplification and analysis of products.

• Do not bring amplified products into the reaction setup area.

• Open and close all sample tubes carefully. Avoid splashing or spraying samples.

• Keep reactions and components capped as much as possible.

• Use a positive-displacement pipettor or aerosol‑resistant barrier pipette tips.

• Clean lab benches and equipment periodically with 10% bleach solution or DNA decontaminationsolution.

Use UNG to prevent false-positive amplificationCarryover amplicons can result in false-positive amplification during PCR. Use a Master Mix thatcontains uracil‑N‑glycosylase (UNG; also known as uracil‑DNA glycosylase (UDG)) to degrade manycontaminating carryover amplicons.

UNG enzymatic activity occurs during an initial incubation at 50°C. UNG is partially inactivated duringthe 95°C incubation step for template denaturation and polymerase activation. Because UNG is notcompletely deactivated during the 95°C incubation, it is important to keep the annealing temperaturesgreater than 55°C and to refrigerate PCR products at 2°C to 8°C in order to prevent amplicondegradation.

To ensure the desired UNG activity:

• Use PCR components and thermal cycling conditions as specified.UNG-containing Master Mixes incorporate the optimal concentration of UNG to prevent cross-contamination while not affecting real-time PCR performance.

• Do not attempt to use UNG-containing Master Mixes in subsequent amplification of dU‑containingPCR products, such as in nested-PCR protocols. The UNG will degrade the dU‑containing PCRproducts, preventing further amplification.

B


Although treatment with UNG can degrade or eliminate large numbers of carryover PCR products, usegood laboratory practices to minimize cross-contamination from non‑dU‑containing PCR products orother samples.

Detect fluorescent contaminantsFluorescent contaminants can generate false positive results. To help detect these contaminants, werecommend including a no‑amplification control reaction that contains sample, but no master mix.

After PCR, if the absolute fluorescence of the no‑amplification control is greater than the fluorescenceof the no template control (NTC), fluorescent contaminants may be present in the sample or in the heatblock of the real-time PCR instrument.

Appendix B Best practices for PCR and RT-PCR experimentsDetect fluorescent contaminants B


Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specified in the userdocumentation may result in personal injury or damage to the instrument or device. Ensure thatanyone using this product has received instructions in general safety practices for laboratories andthe safety information provided in this document.

· Before using an instrument or device, read and understand the safety information provided in theuser documentation provided by the manufacturer of the instrument or device.

· Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and useappropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtainSDSs, see the “Documentation and Support” section in this document.

C


Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnelread and practice the general safety guidelines for chemical usage, storage, and waste providedbelow. Consult the relevant SDS for specific precautions and instructions:

· Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturerbefore you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, seethe "Documentation and Support" section in this document.

· Minimize contact with chemicals. Wear appropriate personal protective equipment when handlingchemicals (for example, safety glasses, gloves, or protective clothing).

· Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only withsufficient ventilation (for example, fume hood).

· Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturercleanup procedures as recommended in the SDS.

· Handle chemical wastes in a fume hood.

· Ensure use of primary and secondary waste containers. (A primary waste container holds theimmediate waste. A secondary container contains spills or leaks from the primary container.Both containers must be compatible with the waste material and meet federal, state, and localrequirements for container storage.)

· After emptying a waste container, seal it with the cap provided.

· Characterize (by analysis if needed) the waste generated by the particular applications, reagents,and substrates used in your laboratory.

· Ensure that the waste is stored, transferred, transported, and disposed of according to all local,state/provincial, and/or national regulations.

· IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposallimitations may apply.

AVERTISSEMENT ! PRÉCAUTIONS GÉNÉRALES EN CAS DE MANIPULATION DE PRODUITSCHIMIQUES. Pour minimiser les risques, veiller à ce que le personnel du laboratoire lise attentive‐ment et mette en œuvre les consignes de sécurité générales relatives à l’utilisation et au stockagedes produits chimiques et à la gestion des déchets qui en découlent, décrites ci-dessous. Consulterégalement la FDS appropriée pour connaître les précautions et instructions particulières à respecter :

· Lire et comprendre les fiches de données de sécurité (FDS) fournies par le fabricant avant destocker, de manipuler ou d’utiliser les matériaux dangereux ou les produits chimiques. Pour obtenirles FDS, se reporter à la section « Documentation et support » du présent document.

· Limiter les contacts avec les produits chimiques. Porter des équipements de protection appropriéslors de la manipulation des produits chimiques (par exemple : lunettes de sûreté, gants ou vête‐ments de protection).

· Limiter l’inhalation des produits chimiques. Ne pas laisser les récipients de produits chimiquesouverts. Ils ne doivent être utilisés qu’avec une ventilation adéquate (par exemple, sorbonne).

· Vérifier régulièrement l’absence de fuite ou d’écoulement des produits chimiques. En cas de fuiteou d’écoulement d’un produit, respecter les directives de nettoyage du fabricant recommandéesdans la FDS.

· Manipuler les déchets chimiques dans une sorbonne.

Appendix C SafetyChemical safety C


· Veiller à utiliser des récipients à déchets primaire et secondaire. (Le récipient primaire contient lesdéchets immédiats, le récipient secondaire contient les fuites et les écoulements du récipient pri‐maire. Les deux récipients doivent être compatibles avec les matériaux mis au rebut et conformesaux exigences locales, nationales et communautaires en matière de confinement des récipients.)

· Une fois le récipient à déchets vidé, il doit être refermé hermétiquement avec le couvercle fourni.

· Caractériser (par une analyse si nécessaire) les déchets générés par les applications, les réactifs etles substrats particuliers utilisés dans le laboratoire.

· Vérifier que les déchets sont convenablement stockés, transférés, transportés et éliminés en res‐pectant toutes les réglementations locales, nationales et/ou communautaires en vigueur.

· IMPORTANT ! Les matériaux représentant un danger biologique ou radioactif exigent parfois unemanipulation spéciale, et des limitations peuvent s’appliquer à leur élimination.

Biological hazard safety

WARNING! Potential Biohazard. Depending on the samples used on this instrument, the surfacemay be considered a biohazard. Use appropriate decontamination methods when working withbiohazards.

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents,and blood of humans and other animals have the potential to transmit infectious diseases.Conduct all work in properly equipped facilities with the appropriate safety equipment (for example,physical containment devices). Safety equipment can also include items for personal protection,such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, orgoggles. Individuals should be trained according to applicable regulatory and company/ institutionrequirements before working with potentially biohazardous materials. Follow all applicable local,state/provincial, and/or national regulations. The following references provide general guidelines whenhandling biological samples in laboratory environment.

· U.S. Department of Health and Human Services, Biosafety in Microbiological and BiomedicalLaboratories (BMBL), 6th Edition, HHS Publication No. (CDC) 300859, Revised June 2020; foundat:https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2020-P.pdf

· Laboratory biosafety manual, fourth edition. Geneva: World Health Organization; 2020 (Laboratorybiosafety manual, fourth edition and associated monographs); found at:www.who.int/publications/i/item/9789240011311

Appendix C SafetyBiological hazard safetyC


https://www.cdc.gov/labs/pdf/CDC-BiosafetyMicrobiologicalBiomedicalLaboratories-2020-P.pdf

https://www.cdc.gov/labs/pdf/CDC-BiosafetyMicrobiologicalBiomedicalLaboratories-2020-P.pdf

https://www.who.int/publications/i/item/9789240011311

Documentation and support

Customer and technical supportVisit thermofisher.com/support for the latest service and support information.

• Worldwide contact telephone numbers

• Product support information– Product FAQs

– Software, patches, and updates

– Training for many applications and instruments

• Order and web support

• Product documentation– User guides, manuals, and protocols

– Certificates of Analysis

– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers, contact themanufacturer.

Limited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forth in theLife Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies atwww.thermofisher.com/support.

D


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http://www.thermofisher.com/support

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